One of the most significant challenges currently facing the field of tissue engineering is the ability to stimulate and/or maintain epithelial cell differentiation in engineered tissues. Since epithelial cell secretory function is crucial to organ function, understanding the mechanisms regulating and maintaining cellular differentiation is critical to regenerating or engineering functional tissues. A man-made functional saliva-secreting salivary gland construct would greatly increase the quality of life for patients suffering from salivary hypofunction, but such engineered tissues have yet to be generated, and in vitro salivary acinar differentiation remains difficult to sustain. The cellular microenvironment plays a significant role in cell differentiation, and yet little is known regarding the specific characteristics of the microenvironment that regulate cell differentiation. Engineered scaffolds often fail to mimic the microenvironment and, in fact, the most effective scaffolds for tissue engineering are decellularized scaffolds derived from live tissue. Since the goal of tissue engineering is to be able to synthesize scaffolds that out-perform decellularized natural scaffolds, it is necessary to understand how the essential characteristics of the natural extracellular matrix (chemical, mechanical/elastic, and topological properties) affect cell differentiation. Recent studies have identified the importance of elasticity of the microenvironment in determining the extent of differentiation of mesenchymal stem cells; however, the significance of elasticity in regulation of epithelial tissue differentiation has not been investigated. Chemical signals, including growth regulatory factors and binding sites, have been much more extensively studied, but the relationship between chemical signals and elasticity remains largely unknown. The overall aim of this project is to define the function of substrate elasticity and cell binding site density in regulating submandibular salivary gland (SMG) acinar cell differentiation. We will use cell lines and embryonic primary cells to address this aim using novel tunable PEG hydrogel scaffolds. We hypothesize that acinar cell differentiation requires a compliant extracellular matrix having optimal cell binding sites which is disrupted at atypical substrate rigidities. To address this hypothesis, we propose to use tunable polyethylene-glycol (PEG)-based hydrogels in three specific aims: Aim 1. Develop PEG-based hydrogels of varied elasticity containing different levels of binding sites. Aim 2. Identify the contributions of elasticity and cell organization in modulating acinar cell differentiation using the hydrogel scaffolds. Aim 3. Use bilayer lithography to create microwell scaffolds for use with primary cells. Abbreviations: AFM, atomic force microscopy; Col IV, collagen type IV; ECM, extracellular matrix; GFP, green fluorescent protein; IKVAV, Isoleucine-Lysine-Valine-Alanine-Valine; PEG, poly(ethylene glycol); PCR, polymerase chain reaction; PEG-DMA, PEG-dimethylacrylate; PEG-TMA, PEG-trimethylacrylate; OMMA, oxiran-2-ylmethyl methacrylate; POMO, 2-((prop-2-ynyloxy)methyl)oxirane; SMG, submandibular salivary gland; transepithelial resistance, TER